Artificially expanded genetic information systems, often known by the acronym AEGIS, have been widely sought [Sis04][Joh04][Liu04][Hen04][Ben04][Ben05], including in the patent literature (U.S. Pat. No. 5,432,272, for “Method for incorporating into a DNA or RNA oligonucleotide using nucleotides bearing heterocyclic bases”; U.S. Pat. No. 6,001,983, for “Oligonucleotides with non-standard bases and methods for preparing same”); U.S. Pat. No. 6,037,120, for “Recognition of oligonucleotides containing non-standard base pairs”; U.S. Pat. No. 6,140,496, for “Precursors for deoxyribonucleotides containing non-standard nucleosides”; and U.S. Pat. No. 5,965,364, for “Method for selecting functional deoxyribonucleotide derivatives”). They are also being mentioned in the popular press [Bal04][Gib04]. A variety of partial solutions to this problem have been reported [Sis04][Del03][Tae01][Hik05].
Without involvement of polymerases, six letter expanded genetic alphabets support clinical assays today that quantitate (through simple Watson-Crick binding) the levels of HIV, hepatitis B and hepatitis C viruses in infected patients; an estimated 400,000 individuals annually benefit in the management of their health care using AEGIS [Elb04a][Elb04b].
The enzymatic synthesis of DNA containing AEGIS components, however, remains problematic [Hor95][Swi93][Swi89], especially when it concerns the amplification of DNA using the polymerase chain reaction. These difficulties are due, in part, to the highly evolved specificity of natural DNA polymerases. This evolution allows them to accept the standard A, G, T, C, but little else with efficiency and high fidelity [Goo93][Mor00][Tab95][Mey04].
Sismour et al. [Sis04] recently reported the PCR amplification of DNA containing a pair between 2,4-diaminopyrimidine and xanthine (called the pyDAD:puADA base pair, because the pyrimidine implements the hydrogen bond Donor-Acceptor-Donor hydrogen bond pattern, from the major groove to the minor groove, complementary to the Acceptor-Donor-Acceptor pattern implemented by the purine xanthine). This was achieved using a double mutant of the reverse transcriptase from human immunodeficiency virus (HIV) I that was obtained by a combination of in clinico selection and rational design. This mutant amplified an oligonucleotide containing a single pyDAD over five rounds of PCR with an overall fidelity (per round) of >99% for the pyDAD:puADA base pair. This process has only narrow utility, however, as the reverse transcriptase is not stable to heating, and therefore must be added anew after each heat cycle.
A second PCR amplification of DNA containing an iso-C:iso-G (pyAAD:puDDA) nucleobase pair was achieved for several dozen rounds of amplification [Jooh04] using a fragment of DNA polymerase I from Thermus aquaticus that does not have an 5′→3′ exonuclease domain, with a fidelity of only ˜96% per round. The T. aquaticus polymerase is stable against thermal denaturation. A fidelity of less than 98% per round is not adequate for most practical applications, including using the six letter alphabet as part of an in vitro selection system.
Similar problems are encountered where steric complementarity is used as the basis for nucleobase pairing specificity [Mor97][Ber00][Hir04].
The loss of fidelity in the system reported by [Joh04] is most likely due to a mispairing of TTP opposite isoG. This mispairing was found in earlier work by Switzer et al. [Swi93]. It is also expected, given the long known fact that isoG has a minor tautomeric form that is complementary to T, and present in aqueous solution to the extent of ca. 10%.